Cell dehydration of intergeneric hybrid induces subgenome‐related specific responses

Abstract The aim was to identify subgenome‐related specific responses in two types of triticale, that is, of the wheat‐dominated genome (WDG) and rye‐dominated genome (RDG), to water stress induced in the early phase (tillering) of plant growth. Higher activity of the primary metabolism of carbohydrates is a feature of the WDG type, while the dominance of the rye genome is associated with a higher activity of the secondary metabolism of phenolic compounds in the RDG type. The study analyzed carbohydrates and key enzymes of their synthesis, free phenolic compounds and carbohydrate‐related components of the cell wall, monolignols, and shikimic acid (ShA), which is a key link between the primary and secondary metabolism of phenolic compounds. Under water stress, dominance of the wheat genome in the WDG type was manifested by an increased accumulation of the large subunit of Rubisco and sucrose phosphate synthase and a higher content of raffinose and stachyose compared with the RDG type. In dehydrated RDG plants, higher activity of L‐phenylalanine ammonia lyase (PAL) and L‐tyrosine ammonia lyase (TAL), as well as a higher level of ShA, free and cell wall‐bound p‐hydroxybenzoic acid, free homovanillic acid, free sinapic acid, and cell wall‐bound syringic acid can be considered biochemical indicators of the dominance of the rye genome.


| INTRODUCTION
Together with the progress of climate change, grows the importance of intergeneric hybrids in breeding projects aimed at obtaining new cultivars or crop species with increased tolerance to environmental stresses, including soil drought (Ali et al., 2020;Rauf et al., 2016;Waqar et al., 2014).
Triticale (Â Triticosecale Witt.), a cross of common wheat (Triticum aestivum L.) and rye (Secale cereale L.) is a classic example of an intergeneric hybrid (Niedziela et al., 2016). This artificially created cereal was supposed to combine the high productivity of wheat and resistance to biotic and abiotic stresses of rye. This objective was achieved by obtaining secondary forms of hexaploid triticale (AABBRR), with wheat (AABB) and rye (RR) genomes (Hura et al., 2009a;Mergoum et al., 2019;Niedziela et al., 2014). Since then, hexaploid triticale has become an interesting model species to investigate the biology of hybrids under environmental stress (Hura et al., 2009b;Szechy nska-Hebda et al., 2015;Wąsek et al., 2022;_ Zur et al., 2013). The investigations include physiological, biochemical, and molecular responses to soil drought and the specific roles the wheat and rye genomes play in these responses Ostrowska et al., 2019). Water stress also identified specific plant properties that result from the interaction of both wheat (AABB) and rye (RR) genomes and that can be uniquely attributed to the triticale genome (AABBRR; Hura et al., 2022).
Earlier studies have shown that the wheat-dominated genome (WDG) in triticale adaptation to water stress in the generative phase is responsible for; for example, higher activity of the photosynthetic apparatus, accumulation of proteins related to photosynthetic fixation of CO 2 or higher level of sugars than in the genotypes with the rye-dominated genome (RDG). Typical responses of the latter to water stress are an increase in the content of cell wall-bound phenolics or the activity of L-phenylalanine ammonia lyase (PAL) and L-tyrosine ammonia lyase (TAL; Hura et al., 2016;Ostrowska et al., 2019). Other adaptive responses of triticale to water stress, visible to the naked eye and indicating the dominance of either genome, maybe the manner of flag leaf rolling or aphid colonization of the flag leaves associated with the content of phenolic compounds in the cell wall structures (Hura et al., 2022). The activity of the triticale genome under water stress, manifested by increased content of cell wallbound phenolics, was confirmed by identification of loci related to this process on 3R and 6R rye chromosomes. These loci harbor serine/threonine kinase and cytokinin oxidase/dehydrogenase 3 genes that may be involved in the complex process of incorporation of phenolic compounds in the cell wall of triticale leaves (Hura, Tyrka, et al., 2017).
So far, the activity of individual triticale genomes under water stress was mainly analyzed during the phase of generative growth, and clear and specific responses of WDG and RDG type due to the activity of either the wheat or the rye genome were confirmed. In addition, specific features and responses resulting from the interplay between these two genomes were identified (Hura et al., 2022).
Therefore, this study aims to analyze specific responses of the wheat and rye genomes during leaf dehydration in the early growth phase of triticale. Such an approach should allow us to select the appropriate growth phase of the intergeneric hybrid for further detailed research on the activity of individual genomes and the interactions between them. We analyzed the primary metabolism related to the synthesis of carbohydrates, the secondary metabolism related to the synthesis of phenolic compounds, and the content of shikimic acid (ShA), which is a key link between the primary and secondary metabolism of phenolic compounds. The intensity of water stress was analyzed by measuring the water potential in leaf cells, the level of abscisic acid (ABA), and stomatal conductance.  (Hura et al., 2022;Hura, Tyrka, et al., 2017).

| Plant growth conditions
The seeds of both triticale types, WDG and RDG, were sown into 3.7 L pots filled with a mixture of soil and sand ( (Hoagland, 1948).

| Soil drought conditions
Soil drought was applied to both triticale types at the stage of four leaves (beginning of tillering). By withholding watering, the water content in the pots was gradually reduced to about 30% for 7 days and kept at this level for the next 14 days (in total 21 days of limited watering). Water content in the control pots was maintained at about 75%. Soil moisture in the pots was inspected daily, between 8.00 a.m. and 10.00 a.m., using a gravimetric method and taking into account plant weight (soil water content in the pots was immediately restored to 30%-drought or 75%-control).

| Measurements and preparation of plant samples
The measurements were performed on Day 21 of the soil drought.
For the analysis, we collected a fully expanded leaf, that is, the fifth one from the bottom. For biochemical and molecular measurements, leaf samples were collected and immediately frozen in liquid nitrogen, lyophilized, and then powdered using stainless steel beads (MM400, Retsch).

| Leaf water potential (Ψ w )
The leaf water potential was analyzed with an HR-33 T dew point microvoltmeter (Wescor, Inc.). Leaf discs (5 mm in diameter) were cut, placed inside the thermocouple psychrometer chamber (C-52) and allowed to reach temperature and water vapor equilibrium for 60 min before measurements were made by the dew point method.

| Protein accumulation
We investigated the proteins responsible for photosynthetic fixation of CO 2 (RbcL-Rubisco large subunit, forms I and II; Agrisera AS03 037) and sucrose phosphate synthase (SPS; global; Agrisera AS03 035A), a protein associated with carbohydrate metabolism. Protein extraction, electrophoresis, western blot, and gel analysis were carried out as described in our previous paper (Hura et al., 2022).

| Carbohydrates
The content of free sugars and starch was analyzed according to Hura et al. (2016). Fructooligosaccharides were estimated following the protocol by Mikuła et al. (2021). HPLC analyses of free sugars, starch, and fructooligosaccharide hydrolysates were done using the Agilent 1200 system (Agilent Technologies) coupled with an ESA Coulochem II 5200A electrochemical detector HPLC (ESA). Separating sugars and starch hydrolysates were done using an RCX-10; 7 μm; 250 Â 4.1 mm column (Hamilton, Ohio) in gradient mode of 75 mM NaOH solution, and 500 mM sodium acetate in 75 mM NaOH solution (Hura et al., 2016).
The average degree of polymerization (DP av ) of the fructans/ oligofructans was calculated using the following formula: DP av = 1  Bradford (1976).
The content of free and cell wall-bound phenolics was estimated according to Hura et al. (2016).

| Monolignols
The samples were extracted as described for phytohormones  and cleaned up in the same manner as for aromatic acids (Hura et al., 2016). D-BeA was added as an internal standard prior to extraction.

| RESULTS
In Table 1, we present changes in water potential (Ψ w ), stomatal conductivity (g s ) and the content of ABA in WDG and RDG triticale exposed to water stress (WS). At a similar level of cell dehydration, ABA content was significantly higher in the WDG type (3827.5 pg mg À1 DW) than in the RDG type (2188.1 pg mg À1 DW).
Under optimal growth conditions, both types showed a similar cell hydration level and ABA content. Stomatal conductivity ( g s ) under water stress conditions was approximately two times lower in WDG than in RDG plants (Table 1).
Western blot analysis indicated higher levels of large Rubisco subunit (RbcL; Figure 1A) and SPS ( Figure 1B) in WDG versus RDG type.
However, water stress (WS) did not evoke any notable changes in the accumulation of these proteins in either triticale type as compared with the control (C).
A significant increase in the activity of PAL (259% of control) and TAL (165% of control) under water stress was only observed for RDG triticale. Water stress did not affect PAL or TAL activity in the WDG type ( Figure 2). Quantitative analyzes of soluble carbohydrates revealed similar and specific changes in their levels ( Our analysis revealed a significant rise in the total amount of fructans in water stress-exposed RDG plants and a significant drop in WDG plants (Table 3). In the RDG type, we detected a significant increase in the average degree of fructan polymerization from 1.51 (C) to 1.59 (WS), while in the WDG type, a reverse pattern was seen, that is, a decrease from 1.80 (C) to 1.56 (WS) ( Moreover, in both types of triticale, a significant reduction in starch content occurred in dehydrated leaves (5.6% of control in WDG, 12.5% of control in RDG) (Table 3).  Table 4).
Quantitative analysis of cell wall-bound phenolics revealed a significant growth in gentisic acid and syringic acid in both triticale types exposed to water stress (WS;  WDG type under water stress (WS; Figure 4). Under the same conditions, no changes in the content of sinapyl alcohol were noted in the WDG type. In RDG plants, water stress induced a significant drop in three analyzed monolignols (56% of control for coniferyl alcohol, 72% of control for sinapyl alcohol, and 65% of control for p-coumaryl alcohol). Under optimal growth conditions (C), WDG and RDG plants significantly differed in their content of coniferyl alcohol (significantly higher content in RDG than in WDG) and p-coumaryl alcohol (significantly lower content in RDG than in WDG), while under water stress, the content of these three monolignols was significantly lower in RDG than in WDG type (Figure 4).

| DISCUSSION
A similar degree of leaf cell dehydration measured based on leaf water potential (Ψ w ), resulted in greater increase in ABA and considerable limitation of stomatal conductance in WDG plants as compared with RDG ones (Table 1). Accumulation of ABA is a marker of stress intensity (Wang et al., 2022). The level of ABA found in our experiment confirmed the involvement of the rye genome in water stress tolerance (Mubarik et al., 2021). However, it was shown in some species that ABA treatment does not always improve drought tolerance (Bartels & Salamini, 2001;Wu et al., 2020). Other studies also demonstrated that translocation of rye chromosome segment 1RL and 1RS into wheat enhances soil drought tolerance, benefits yielding, and improves water use efficiency by promoting root and aboveground biomass growth (Ehdaie et al., 2003;Hoffmann, 2008;Karki et al., 2014).
Although water stress did not affect the content of proteins responsible for photosynthetic fixation of CO 2 and carbohydrate synthesis in WDG and RDG plants, the WDG type accumulated considerably greater amounts of the larger subunit of Rubisco (RbcL; Figure 1A) and SPS ( Figure 1B). These results contradict our previous findings on the induction of water stress in the vegetative growth phase of triticale, in which the accumulation of SPS and RbcL was clearly lower in the dehydrated leaves (Hura et al., 2018). The dominance of the wheat genome in the WDG type was evident under optimal growth conditions and it was manifested by significantly higher levels of the analyzed carbohydrates, such as glucose, sucrose, maltose, raffinose, 111-5-kestose and nystose, compared to the RDG type ( Table 2). The analyses were performed during tillering at the time of intense cell divisions (Yang et al., 2022). Assuero et al. (2012) showed that an increased carbohydrate content in wheat plants might lead to faster leaf and tiller development. Our results for the WDG hybrid indicate that carbohydrate level may be an additional marker for the selection of genotypes with increased tillering potential or productivity associated with high levels of soluble carbohydrates, which may translate into enhanced yielding under optimal growth conditions (Saint Pierre et al., 2010).
Lower carbohydrate content (sucrose, maltose, raffinose, kestose, and 111-5-kestose, nystose) in the WDG type under water stress accompanied by accumulation of proteins responsible for carbohydrate synthesis, RbcL, and SPS, may be due to intense consumption of carbohydrates during water stress (Raineri et al., 2015). In the same conditions, the activity of the wheat genome in the WDG type was manifested by a significantly increased content of raffinose and stachyose as compared with the RDG type (Table 2). Raffinose and stachyose are key osmolytes that protect cell components and maintain the osmotic balance in wheat (Kerepesi & Galiba, 2000). Similarly, as in wheat, dos Santos et al. (2011) showed a protective role of raffinose and stachyose accumulation in Coffea arabica leaves exposed to osmotic stress. These carbohydrates are also suggested to participate in the scavenging of reactive oxygen species (ROS;Peshev et al., 2013), transport and storage of carbon (Elsayed et al., 2014), mRNA export (Okada & Ye, 2009), membrane trafficking (Thole & Nielsen, 2008), signaling (Stevenson et al., 2000;Xue et al., 2007), as well as stabilizing photosystem II (Knaupp et al., 2011) or other proteins (Bartels & Sunkar, 2005). It should be emphasized that other studies showed accumulation of carbohydrates not only in droughttolerant plants but also in drought-sensitive ones (Nemati et al., 2018;Ozturk et al., 2021).
At the same time, a low DP for fructans and oligofructans (1 < DP <2) indicates that the synthesis of low-molecular weight fructans and oligofructans dominates in the leaves of both triticale types, irrespective of growth conditions (Table 3) of carbohydrates but also the types of synthesized carbohydrates (Gilbert et al., 1997). Other recent studies based on transcriptomic, proteomic, and metabolomics approaches showed involvement of primary carbon metabolism in plant responses to water stress (Jiang et al., 2021;Kumar et al., 2021).
ShA is an important intermediate in the biosynthesis of secondary metabolites, such as phenolic compounds, involved in plant adaptation to water stress (Hura et al., 2016;Scalabrin et al., 2016). In our study, the decreased level of ShA in WDG plants ( Figure 3) indicates a weaker activation of plant defense mechanisms than in RDG type. On the other hand, the increase in ShA content in water stress-exposed RDG plants could be related to the mobilization of secondary metabolism (Scalabrin et al., 2016). Sicher and Barnaby (2012) also reported about a 10-fold increase in ShA accumulation under water stress in maize. Other observations were reported for maize (Li et al., 2021) and oil tea (Qu et al., 2019), where plants grown under water stress showed lower levels of ShA.
The metabolism of phenolic compounds was analyzed based on the activity of PAL and TAL. Their activity is affected by environmental stresses (Huang et al., 2010), including water stress (Hura et al., 2009b). A significant increase in the activity of both enzymes in dehydrated leaves was determined only in RDG plants (Figure 2), which indicated a special role of the rye genome in the metabolism of phenolic compounds. This was confirmed in another report (Hura, Tyrka, et al., 2017), which showed that the loci associated with the incorporation of phenolic compounds into the cell wall structures under soil drought are located mainly on rye chromosomes, that is, one locus on Chromosome 3R and two loci on Chromosome 6R.
Under the same conditions, in wheat, only 1 locus was located on Chromosome 4B.
The importance of the rye genome in controlling secondary metabolism, analyzed by quantifying phenolic compounds, has been confirmed for several substances. As for soluble phenolic compounds, the RDG type accumulated more p-hydroxybenzoic acid, homovanillic acid, and sinapic acid than the WDG type, both under optimal and limited hydration. Moreover, under optimal conditions, the RDG type accumulated more cinnamic acid (Table 4). These findings may suggest a role of the rye genome in the selective activation of pathways of phenolic compound synthesis that results in the accumulation of specific phenolics (Hura et al., 2016). Enhanced accumulation of phydroxybenzoic acid, homovanillic acid, and sinapic acid under water stress was also reported in previous studies Quan & Xuan, 2018), including those in triticale (Hura et al., 2016). Recent studies underline the significance of individual phenolic compounds for plant defense mechanisms under water stress conditions (Kravic et al., 2021;Sarker & Oba, 2020).
RDG plants showed higher p-hydroxybenzoic acid and syringic acid content in the cell wall structures than WDG plants (Table 4). Horváth et al., 2002 reported that p-hydroxybenzoic acid increases the impermeability of cell walls, which can limit water loss in plants exposed to water stress (Hura et al., 2016;Hura et al., 2022). It has also been suggested that the increase in the content of phenolic compounds in the cell wall may be due to their inability to form stable bonds with glucose (glucoside synthesis) in the cytosol. This seems to be associated with the low activity of glucosyltransferases specific for individual phenolics (Meyermans et al., 2000). Therefore, the more intense incorporation of these substances into the cell wall structures of the RDG type could result from the limited synthesis of glucosides from p-hydroxybenzoic acid/syringic acid and glucose in the cytosol than in the WDG type.
Monolignols are secreted into the cell wall and cross-linked through oxidative polymerization. The cross-linking depends on the availability of ROS generated, among others, by the cell wall peroxidases. This process reinforces the strength and rigidity of cell walls and can be a crucial element of plant response to environmental factors, including water stress (Le Gall et al., 2015). However, our study showed mainly a significant decrease in coniferyl alcohol, sinapyl alcohol, and p-coumaryl alcohol under water stress (Figure 4). This significantly lower level of the three monolignols in RDG and p-coumaryl alcohol in WDG as compared with well-watered plants could be explained by increased cell wall cross-linking in this area under water stress (Grabber et al., 2000;Vincent et al., 2005). This way, the cell wall saturated with phenolic compounds improves plant tolerance to water stress (Liu et al., 2018).
In conclusion, water stress in the vegetative growth phase of the intergeneric hybrid induced specific physiological and molecular responses related to the dominance of the wheat genome in the WDG type and the rye genotype in the RDG type. The dominance of the wheat genome is mainly manifested by higher activity of the primary metabolism assessed by the level of carbohydrates, while the dominance of the rye genome is related to, among other things, higher activity of the secondary metabolism of phenolic compounds.
In the WDG type, the higher accumulation of RbcL and SPS, as well as the increase of some carbohydrates, can be considered biochemical and molecular indicators of the wheat genome dominance in nonstressed plants. Under stress, the WDG type showed only increased accumulation of RbcL and SPS and a higher content of raffinose and stachyose than the RDG type. In the RDG type, the indicators of the rye genome dominance in nonstressed plants were the increased content of ShA, some free and cell wall-bound phenolics, and coniferyl alcohol. In dehydrated RDG plants, the dominance of the rye genome was manifested by a higher than in WDG plants activity of PAL and TAL, as well as a higher level of ShA, free and cell wall-bound p-hydroxybenzoic acid, free homovanillic acid, free sinapic acid, and cell wallbound syringic acid. It should be emphasized that water stress in the early growth phase did not induce as strong specific and differentiating responses of the wheat and rye genomes as it did in the generative growth phase of triticale (Hura et al., 2022). Nonetheless, we suggest that both growth phases of this intergeneric hybrid are suitable for further studies on the precise role of the wheat and rye genomes and the identification of specific features and responses resulting from the interaction between these genomes under water stress.

DATA AVAILABILITY STATEMENT
The data that support the findings are available from the corresponding author upon reasonable request.